Animals are not known to biosynthesize floral chemical signals to manipulate pollinators, although such mimicry could profoundly shape plant-pollinator interactions. Larvae of the poisonous European blister beetle Meloe proscarabaeus parasitize multiple solitary bee species, yet the mechanism enabling host attraction has remained unresolved. Here we show that these larvae lure bees by emitting a bouquet of volatile compounds that closely resembles floral scent. Chemical analyses reveal a complex blend of monoterpenoids derived from (S)-linalool, a ubiquitous floral volatile. Behavioral assays demonstrate that these compounds function as floral-scent mimics, eliciting attraction in bees and acting as allomones (i.e., interspecific chemical signals that benefit the emitter while disadvantaging the receiver). Transcriptomic and functional analyses identify cytochrome P450 enzymes that oxidize (S)-linalool, suggesting that larvae biosynthesize these plant-like volatiles de novo. Together, these findings broaden the scope of interkingdom chemical mimicry and uncover a striking form of sensory deception in which an insect chemically assumes the signal identity of a flower, revealing that animals can evolve biosynthetic pathways to exploit plant-pollinator communication.

Halogenation of natural products can improve their bioactivity, pharmacokinetics, and commercial value, yet sustainable production of halogenated small molecules remains challenging. Benzoxazinoids (BXDs) are indole-derived plant specialized metabolites whose halogenated derivatives possess promising pharmaceutical and agrochemical properties. Here, we engineered Nicotiana benthamiana to produce halogenated BXDs through both precursor-directed and de novo biosynthetic strategies. Screening 22 BXD biosynthetic enzymes from diverse plant species identified a subset of enzymes with substrate promiscuity toward halogenated indole analogs, enabling the formation of chlorinated and fluorinated BXDs in transiently transformed N. benthamiana plants supplied with halogenated precursors. To establish a fully plant-based production pathway, we co-expressed this enzyme set with a biosynthetic module that provides chlorinated indole analogs and includes bacterial tryptophan halogenases, a bacterial flavin reductase, and a bacterial tryptophanase. This strategy resulted in the de novo production of substantial amounts of chlorinated BXDs in planta. Furthermore, chlorinated BXDs could be converted into chlorinated benzoxazolinone analogs, compounds with pharmaceutical relevance. These findings demonstrate that N. benthamiana provides a versatile and sustainable platform for the biosynthesis of halogenated BXDs and related high-value halogenated natural products.

Indole is an important biomolecule in plants, essential for amino acid biosynthesis, defense, pollinator attraction and plant-plant communication. Its biosynthesis is reported to be catalyzed by standalone indole-3-glycerol phosphate lyases, which are, however, absent in core eudicots. Here we show that, in core eudicots, indole production for defense and signaling occurs through an alternative pathway. The tryptophan synthase α subunit (TSA), which is typically complexed with the β subunit (TSB) to synthesize tryptophan through indole as an intermediate, can be hijacked by a noncatalytic paralog of TSB (TSB-like) to produce free indole. TSB-like is a pseudoenzyme that evolved from TSB by mutagenesis of two key essential residues, retaining the ability to allosterically activate TSA to allow formation and release of indole. The widespread occurrence and expression pattern of TSB-like genes in plants suggest that this is a general mechanism for the formation of free indole in plant defense and communication.

After consumption by herbivores, plant antimicrobial defense compounds may enhance herbivore immunity to pathogenic microbes. In conifer-bark beetle interactions, beetles ingest large quantities of phloem tissue containing high concentrations of antimicrobial phenolic glucosides, such as stilbenes and flavonoids. It is not known, however, if these compounds increase bark beetle resistance to pathogens. We showed that Eurasian spruce bark beetles (Ips typographus) attacking Norway spruce (Picea abies) hydrolyze phenolic glucosides to their corresponding aglucones increasing their antifungal activity. However, the entomopathogen Beauveria bassiana, a natural fungal parasite of these beetles, detoxifies stilbene and flavonoid aglucones by forming methylglucoside derivatives. A two-step pathway involving a UDP-glycosyltransferase and an O-methyltransferase produces phenolic O-methylglucosides that are no longer toxic to B. bassiana and are stable to β-glucosidase action. Compared to wild-type strains of B. bassiana, mutant strains knocked out in the genes of this pathway exhibited decreased methylglucoside formation, slower growth on medium containing phenolic compounds, and reduced virulence toward bark beetles. Hence, methylglucosylation of plant-derived phenolics is a detoxification process that significantly increases the ability of B. bassiana to parasitize host insects consuming plant tissue high in phenolics, such as conifer phloem. This is one of the few examples of an entomopathogen that is able to resist the plant-derived defenses of an insect host.

Brassicales plants defend themselves with glucosinolates that, upon herbivory, are hydrolyzed into toxic isothiocyanates (ITCs) and other derivatives. The side chain diversity of glucosinolates results in a range of structurally distinct products, but how this chemical variation affects herbivores and their detoxification responses remains incompletely understood. Here, we show the effects of ITC hydrolysis products with various side chains on Spodoptera littoralis larvae and their detoxification system. ITCs inhibit larval growth to varying degrees, depending on the chemical nature of their side chain. The larvae metabolize ITCs by conjugating them to glutathione in the mercapturic acid pathway and to lysine forming an amine conjugate. Over half of the 34 S. littoralis glutathione-S-transferases (GSTs), tested as His-tagged derivatives, actively conjugate ITCs, with most catalyzing reactions with multiple substrates. Larval performance on various ITC-containing diets correlates positively with GST activity, highlighting this detoxification system's role in supporting growth on glucosinolate-containing plants. The propensity of multiple GSTs to react with an individual ITC and the wide expression of GST-encoding genes across larval organs likely promote the ability of this generalist herbivore to thrive on glucosinolate-defended Brassicales plants. These findings provide insight into herbivore adaptation and may inform future research on plant-insect interactions.

Iridoids are bicyclic monoterpenoids that function as defense and signaling compounds in both plants and insects. Although recent research suggested that iridoids evolved independently in these two kingdoms of life, it remained unclear whether independent evolution of iridoid biosynthesis also occurred across different insect lineages. In order to gain insight into the evolution of iridoids in insects, we examined the first committed step of iridoid biosynthesis, the hydroxylation of geraniol to 8-hydroxygeraniol, in the Argentine ant (Linepithema humile) of the order Hymenoptera. This transformation is typically catalyzed by cytochrome P450 monooxygenases in both plants and insects. By integrating transcriptomic and metabolomic analyses across various body parts, we identified candidate P450 genes potentially involved in this reaction. The candidate genes were heterologously expressed in yeast, and enzyme activity was assessed by supplying geraniol to the yeast cultures. One candidate P450 enzyme displayed geraniol 8-hydroxylase (G8H) activity and was designated LhG8H. Phylogenetic analysis showed that LhG8H is evolutionarily distinct from previously characterized G8H enzymes in the pea aphid (Acyrthosiphon pisum) of the order Hemiptera and the mustard leaf beetle (Phaedon cochleariae) of the order Coleoptera. These results support the hypothesis that geraniol 8-hydroxylase activity, a key step in iridoid biosynthesis, evolved independently within the insect orders Hymenoptera, Hemiptera, and Coleoptera.

Plant-specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. Monoterpene indole alkaloids (MIAs) tabersonine and pseudo-tabersonine, found in the medicinal plant Tabernanthe iboga (commonly known as iboga), are tailored with oxidations, and the enzymes involved remain unknown. Here, we developed a streamlined screening strategy to test the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo-tabersonine scaffolds, we aimed to uncover the CYP450s responsible for oxidative transformations in these scaffolds. Our approach identified two T. iboga cytochrome P450 enzymes: pachysiphine synthase (PS) and 16-hydroxy-tabersonine synthase (T16H). These enzymes catalyze an epoxidation and site-specific hydroxylation of tabersonine to produce pachysiphine and 16-OH-tabersonine, respectively. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and Catharanthus roseus as platforms for the functional characterization of plant enzymes.

Here, we use transcriptomic data from seeds of Musella lasiocarpa to identify five enzymes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to phenylphenalenones and linear diarylheptanoid derivatives, the major seed metabolites. Furthermore, we demonstrate the stepwise conversion of dihydrobisdemethoxycurcumin to the phenylphenalenone 4 '-hydroxylachnanthocarpone by feeding intermediates to M. lasiocarpa root protein extract.

Indole is crucial for plant defense, where it is released as a signaling volatile upon herbivore attack and also serves as a starting precursor for defensive specialized metabolites. Indole is known to be synthesized in plants from indole-3-glycerol phosphate by the enzyme indole-3-glycerol phosphate lyase. Here we report that in core eudicots, indole production for plant defense occurs via an alternative pathway. The α subunit of tryptophan synthase (TSA), an enzyme of core metabolism, normally binds to tryptophan synthase β subunit (TSB) to produce tryptophan. However, we show that a non-catalytic TSB paralogue (TSB-like) can highjack TSA to produce indole. The widespread occurrence of TSB-like genes in eudicots suggests that this alternative mechanism for indole formation is widespread throughout the plant kingdom. ### Competing Interest Statement The authors have declared no competing interest.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Beauveria bassiana is a soil fungus that parasitizes a large number of arthropod species, including numerous crop pests, causing white muscardine disease and is therefore used as a biological insecticide. However, some insects, such as the cabbage aphid (Brevicoryne brassicae), defend themselves chemically by sequestering dietary pro-toxins (glucosinolates) from their Brassicales host plants. Glucosinolates are accumulated by cabbage aphids and activated to form toxic isothiocyanates when under attack. While isothiocyanate formation protects aphids against most attackers, B. bassiana is still able to infect the cabbage aphid under natural conditions. We therefore investigated how this fungus is able to circumvent the chemical defense system of the cabbage aphid. Here, we describe how B. bassiana infection activates the cabbage aphid defense system, but the resulting toxins are metabolized by B. bassiana via the mercapturic acid pathway, of which the first step is catalyzed by glutathione-S-transferases of low substrate specificity. This detoxification pathway enhances B. bassiana growth when isothiocyanates are present in natural concentrations, and so appears to be an important factor in fungal parasitization of these chemically defended aphids.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra- and inter-specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing-sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS-type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions.

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

Background Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp . Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar ( P. nigra ) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. Results We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTI s via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTI s than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. Conclusions We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Plant volatiles play a major role in plant–insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant–insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar ( Populus nigra ) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend . For one endophyte, a species of Cladosporium , we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like ( E )-β-ocimene and ( E )-β-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene ( E )-β-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore.